title: Endo-PDI is required for TNFα-induced angiogenesis.
abstract: Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function.
authors.affiliation: Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Livia de Lucca
authors.initials: LL
authors.lastname: Camargo
authors.affiliation: Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Babelova
authors.affiliation: Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Mieth
authors.affiliation: Institute for Biochemistry I, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Weigert
authors.affiliation: Institute for Biochemistry II, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Mooz
authors.affiliation: Institute for Biochemistry II, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Krishnaraj
authors.initials: K
authors.lastname: Rajalingam
authors.affiliation: Functional Proteomics, SFB815 Core Unit, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Heide
authors.affiliation: Functional Proteomics, SFB815 Core Unit, Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Wittig
authors.affiliation: Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Lucia Rossetti
authors.initials: LR
authors.lastname: Lopes
authors.affiliation: Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany. Electronic address: r.brandes@em.uni-frankfurt.de.
authors.forename: Ralf P
authors.initials: RP
authors.lastname: Brandes
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology*; D006801:Humans; D020672:I-kappa B Proteins; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis; D019255:NADPH Oxidases; D000072000:NF-KappaB Inhibitor alpha; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010766:Phosphorylation; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D034622:RNA Interference; D034741:RNA, Small Interfering; D018874:Spheroids, Cellular; D013880:Thioredoxin-Disulfide Reductase; D018808:Transcription Factor AP-1 / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics
chemical_list: D043925:Angiogenesis Inducing Agents; D015815:Cell Adhesion Molecules; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C533912:CTSB protein, human; D002401:Cathepsin B; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9; D018631:ras Proteins; D019704:Protein Disulfide-Isomerases; C480743:TXNDC5 protein, human
keywords: AP-1; ER; ERK1/2; Endothelium; Free radicals; Inflammation; MAK kinase; MAP kinase; MAP kinases; MEK; MMP; NADPH oxidase; PDI; TNFα; Thiol; endoplasmic reticulum; extracellular-regulated kinase; matrix metalloproteinase; mitogen-activated protein kinase; protein disulfide isomerase; transcription factor activator protein-1; tumor necrosis factor α